T cells play a key role in the regulation of immune responses and are important for establishing immunity to pathogens. In addition, T cells are often activated during inflammatory autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, type I diabetes, multiple sclerosis, Sjogren's disease, myasthenia gravis, psoriasis, and lupus. T cell activation is also an important component of transplant rejection, allergic reactions, and asthma.
T cells are activated by specific antigens through the T cell receptor (TCR) which is expressed on the cell surface. This activation triggers a series of intracellular signaling cascades mediated by enzymes expressed within the cell (Kane, L P et al. Current Opinion in Immunol. 200, 12, 242). These cascades lead to gene regulation events that result in the production of cytokines, like interleukin-2(IL-2). IL-2 is a critical cytokine in T cell activation, leading to proliferation and amplification of specific immune responses.
One class of enzymes shown to be important in signal transduction is the kinase proteins. PKC enzymes are members of a distinct family of serine/threonine protein kinases that contain nine members (isotype α, β, γ, δ, ε, ζ, η, θ, ι)(reviewed in Nishizuka Y., Science 1992; 258:607-614), some of which are expressed at particular high levels in T cells (including α, δ, ε, η, θ)(reviewed in Baier, G., Immunological Reviews 2003 192:64-79). Gene disruption studies suggest that inhibition of some members of the PKC family of kinases would potentially lead to therapeutic benefit. PKCα (−/−) mice and mice deficient in PKCθ both have T cell defects (Baier, G., Immunological Reviews 2003 192:64-79; Pfeifhofer C. et. al, Journal of Experimental Medicine, 197:1525-1535;Sun, Nature 2000, 404:402-407), suggesting that inhibition of either of these kinases would be useful in diseases of T cell mediated inflammation and autoimmunity. PKCθ in particular may be a prime target for novel anti-inflammatory or immuno-suppressive therapies, due to its restricted tissue-expression and its nonredundant critical role in TCR-mediated IL-2 secretion (N. Isakov and A. Ammon Annu. Rev. Immunol. 2002 20:761-94). Small molecule drugs selectively inhibiting PKCθ and/or other certain other PKC isoenzymes such as PKC alpha, beta, epsilon and zeta may manifest improved efficacy and/or improved side-effect profile over drugs targeted against other immune-mediators suc has calcineurin and Akt1/PKBalpha. For example, a dual inhibitor of both PKC theta and PKC alpha may effectively prevent mature T cell activation.
PKC alpha, like PKC theta, is involved in TCR signaling in T cells (Iwamoto 1992 JBC 267:18644-18648; Ohkusu 1997 J. Immunol. 159:2082-2084). PKC family kinases are also important for signaling downstream of other immune cell receptors. PKC beta participates in B cell receptor signaling (Leitges M. et al. 1996Science 273:788-791), neutrophils (Dekker L V et al. 2000 Biochem. J. 347:285-289), and mast cells (Nechushian H et al. 2000 Blood 95:1752-1757). PKC zeta also plays a role in B cell signaling and function (Martin P. et al. 2002 EMBO J. 15:4049-4057) and PKC epsilon is required for macrophage activation (Castrillo A. et al. 2001 J. Exp. Med. 194:1231-1242). These findings suggest that PKC family kinase inhibitors may be useful in treating inflammatory, autoimmune and allergic diseases and asthma.
In addition to its essential function in mature T cell activation and IL-2secretion, PKC theta provides a survival signal that protects leukemic T cells from Fas-ligand induced apoptosis (M. Villalba and A. Altman 2002 Current Cancer Drug Targets 2:125-134). This feature and the constitutive membrane location of PKC theta in some leukemic T cells suggest that it plays a role in the growth and survival of leukemic T-cells. Furthermore, the high-affinity IL2 receptor (IL-2R alpha) is constitutively expressed by some malignant T cell leukemias suggesting that expansion of these cells may be supported by an IL-2 autocrine loop (M. Villalba and A. Altman 2002 Current Cancer Drug Targets 2:125-134). PKC theta may also promote survival of malignant cells by functioning in development of a multidrug resistance (MDR) phenotype. PKC theta expression is positively correlated with the expression of some genes involved in MDR including MDR1and MRP1 in acute myelogenous leukemia patients (Beck J. et al. 1996 Leukemia 10:426-433) and PKC theta regulates MDR1 promoter activity in human breast carcinoma cells (Gill P. K. et al. 2001 Eur. J. Biochem 268:4151-4157). Therefore, a PKC theta small molecule inhibitor may facilitate elimination of leukemic T cells and other malignant cells that over-express PKC theta. Concomitant overexpression/activation of both PKC alpha and PKC theta has also been implicated in development of multi-drug resistance. Therefore a dual PKC theta and PKC alpha small molecule inhibitor may also facilitate elimination of malignant cells that overexpress both PKC alpha and PKC theta.
Other groups have published on inhibitors of PKC family kinase and the activities of these inhibitors in various in vitro and in vivo biological systems. For example, PCT Publication No. WO 2004067516 discloses 2,4-diaminopyrimidine derivatives useful as inhibitors of PKC-theta. WO 2003082859 discloses indolylmaleimide derivatives as compounds useful in the treatment and/or prevention of diseases or disorders mediated by T-lymphocytes and/or PKC. The protein kinase C beta inhibitor ruboxistaurin (LY-333531), the lead compound from a series of 14-membered macrocycles, is being developed for the potential treatment of diabetic retinopathy, diabetic macular edema and diabetic neuropathy (Investigational Drug database, Dec. 19, 2003, Ruboxistaurin update). By October 2003, this compound was also being investigated as a potential treatment for cardiovascular disease in diabetic patients. It was in phase III trials for both diabetic retinopathy and macular edema by early 2001.